Effect Of Cytokines In Host Defense And Inflammation

今年，我们继续在人类捐赠者身上使用皮肤吸引水泡的无菌炎性渗出物模型。在过去的六个月里，我们招募了9名捐赠者，并建立了基线细胞因子反应，部分复制了本实验室以前的结果，但也识别了以前没有报道过的在水泡形成过程中上调的细胞因子。我们正计划使用这种水泡技术来探索免疫功能障碍患者的炎症反应，以及局部使用免疫调节剂治疗的正常患者的炎症反应。我们已经同时从渗出细胞和外周细胞中收集了RNA，并将使用微阵列技术来识别滞育诱导的基因组调节。 我们继续研究纤维蛋白原作为IL8产生的调节因子。在以前的工作中，我们发现纤维蛋白原能增强中性粒细胞在其他化学诱导剂如fmetleuphe和LTB4刺激下合成IL8。我们将这项研究扩展到人类单核细胞，结果表明，添加生理浓度的纤维蛋白原可以增强单核细胞产生IL8，并增加IL6和肿瘤坏死因子α的产生。相反，纤维蛋白原对单核细胞趋化蛋白1(MCP1)、干扰素β和干扰素诱导蛋白10(IP10)无影响。单核细胞经纤维蛋白原(小于2毫克/毫升)和补体5片段C5a处理后，IL-8和IL-6的产生均较单独纤维蛋白原处理增加100%。这与单核细胞IL8mRNA和NFkB活性的一过性增加有关。与正常单核细胞相比，通过Tolllike受体途径(NEMO缺乏和IRAK4缺乏)的内毒素和IL1信号缺陷患者的单核细胞对纤维蛋白原的反应降低了80%。此外，正常单核细胞对纤维蛋白原的反应可被阻断CD14的抗体阻断，CD14是脂多糖受体的一个亚单位，通过TLR4传递信号。MY4对PMA和离子霉素诱导的细胞因子的产生没有影响。(面团库恩斯) 我们继续研究Toll样受体途径障碍和反复细菌感染(IRAK4缺乏症和NEMO缺乏症)患者启动和激活NADPH氧化酶和超氧化物歧化酶的分子基础。今年，我们将这些研究的重点放在内毒素(LPS)产生超氧化物的中性粒细胞上。在正常人中，中性粒细胞单独与脂多糖孵育可诱导NADPH氧化酶胞浆因子p47Phox和rac2，以及膜整合亚基gp91Phox移位到膜上。与单独加入内毒素或fMLP相比，加入fMLP可增加中性粒细胞NADPH氧化酶调节组分(p47Phox、p67Phox、p40Phox、rac2和gp91Phox)的易位。磷脂酰肌醇3激酶(PI3K)抑制剂LY2940002显著抑制了脂多糖对p47Phox转位和超氧化物生成的启动作用。我们还观察到IRAK4缺乏症患者中性粒细胞在脂多糖作用下产生的超氧化物歧化，这与胞浆因子(p47Phox、p67Phox、p40Phox、rac2)和膜整合黄细胞素b558亚单位gp91Phox的低效转位有关。然而，NEMO缺乏症患者的中性粒细胞比IRAK4缺陷症患者的中性粒细胞对脂多糖启动的反应更快，氧化酶调节亚基的易位水平正常。与正常细胞和NEMO缺陷细胞相比，我们注意到IRAK4缺陷细胞的中性粒细胞中p47Phox的不完全磷酸化。然而，与TLR信号缺陷患者中性粒细胞不同的启动和激活不同，IRAK4和NEMO缺陷细胞中TLR的表达水平保持不变。与IRAK4缺陷细胞一样，LY2940002的使用导致超氧化物生成水平低于正常水平，以及p47Phox的最低磷酸化和易位。(安加利·辛格)

This year we continued to use a model of sterile inflammatory exudates in human donors using skin suction blisters. Over the past six months we have enrolled 9 donors and have established a baseline cytokine response in part reproducting previous results from this lab, but also identifying additional cytokines not previously reported to be upregulated during blister formation. We are planning on using this blister technique to explore inflammatory responses in patients with immune dysfunction but also in normal patients treated topically (at the blister site) with immunomodulatory agents. We have collected RNA concurently with exudate cells and from peripheral cells and will be using microarray technology to identify genomic regulation induced by diapediesis.(Kol Zarember) We continued our studies of fibrinogen as a regulator of IL8 production. In previous work we showed that fibrinogen amplifies IL8 synthesis in neutrophils stimulated with other chemoattractants such as fmetleuphe and LTB4. We extended this studies to human monocytes and showed that addition of physiological concentrations of fibrinogen amplified IL8 production by monocytes as well as increased IL6 and TNF alpha production. In contrast, fibrinogen had no effect on monocyte chemoattractant protein1 (MCP1), inteferonbeta, or interferon inducible protein10 (IP10). Treatment of monocytes with fibrinogen (less than 2 mgml) and complement 5 fragment, C5a, resulted in a 100% increase in both IL8 and IL6 production, compared to fibrinogen treatment alone. This was associated with a transient increase in monocyte IL8 mRNA and NFkB activity. Monocytes from patients with defective LPS and IL1 signaling through the Tolllike receptor pathway (NEMO deficiency and IRAK4 deficiency)had 80% reduced IL8 response to fibrinogen compared with normal monocytes. Moreover, normal monocyte responses to fibrinogen were blocked by antibody that blocks CD14, a subunit of the LPS receptor that transduces signal throug TLR 4. MY4 had no effect on cytokine production induced by PMA and ionomycin. (Dough Kuhns) We continue to study the molecular basis for priming and activation of the NADPH oxidase and superoxide in patients with disorders of the Toll like receptor pathway and recurrent bacterial infections (IRAK4 deficiency and NEMO deficiency). This year we focused these studies on neutrophil priming for superoxide production by endotoxin (LPS). In normal subjects incubation of neutrophils with LPS alone induced NADPH oxidase cytosolic factors p47phox and Rac2, and the membrane integrated subunit gp91phox translocation to the membrane. Addition of fMLP to LPS treated neutrophils augmented the translocation of NADPH oxidase regulatory comonents (p47phox, p67phox, p40phox, Rac2 and gp91phox) compared with LPS or fMLP alone. LPS mediated phosphorylation of p47phox ran parallel to the priming effect of LPS on p47 phox translocation and superoxide production, though this was significantly inhibited by the phosphatidylinositol 3kinase (PI3K)inhibitor LY2940002. We also observed impaired superoxide generation by LPS in neutrophils from patients with IRAK4 deficiency that correlated with the inefficient translocation of cytosolic factors (p47phox, p67phox, p40phox, Rac2) and membrane integrated flavocytochrome b558 subunit gp91phox. However, neutrophils from patients with NEMO deficiency were much more responsive to the LPS priming than IRAK4 deficient cells, with normal levels of translocation for the oxidase modulatory subunits. Compared to the normal cells and NEMO deficient cells, we noted the incomplete phosphorlyation of p47phox in neutrophils of IRAK4 deficient cells. However, unlike the diferential priming and activation in the neutrophils from patients with TLR signaling defects, the levels of TLR expression remained unchanged in both IRAK4 and NEMO deficient cells. The use of LY2940002, as with IRAK4 deficient cells, led to the sub normal levels of superoxide generation and minimalphosphorylation and translocation of p47phox. (Anjali Singh)